Bi-specific antibody engagers for cancer immunotherapy

Bispecific antibody engagers are fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains (VHHkappa) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumor activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. In the MC38 mouse model of colorectal carcinoma, the anti-CTLA-4 VHH-VHHkappa conjugate eradicates tumors and reduces the number of intratumoral regulatory T cells. The anti-PD-L1 VHH-VHHkappa conjugate is less effective in the MC38 model, whilst still outperforming an antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHHkappa conjugate was strongly enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype.


Introduction
Antibodies exert their protective and therapeutic effects by different mechanisms. 1 Ligation of surface receptors by antibodies can either activate or inhibit the targeted structure.By occluding features essential to a protein's function, antibodies can block receptor-ligand interactions, impede access to substrates or thwart a pathogen's route of access to the host.The variable regions of the immunoglobulin (Ig) heavy and light chains account for the speci city of recognition.An antibody's effector functions, carried in its Fragment crystallizable (Fc) region, enable cytotoxicity that is either complement-mediated (complement-dependent cytotoxicity; CDC) or carried out by cells that bear Fc receptors (FcR) of the appropriate speci city (antibody-dependent cell-mediated cytotoxicity; ADCC).Phagocytosis of Ig-decorated structures such as viruses, bacteria or cell remnants is likewise mediated by Fc receptors (antibody-dependent cell-mediated phagocytosis; ADCP). 2,3erapeutic antibodies are typically deployed as a single isotypic variant, such as an IgG1 or IgG4. 4 Features that allow engagement of Fc receptors may be inactivated by introduction of suitable mutations or left intact, depending on the mechanism of action of the therapeutic agent. 3The Fc portions of the different Ig isotypes show distinct activities with respect to complement activation and Fc receptor engagement, resulting in different outcomes: whereas the Fc portion of an IgE molecule is particularly good at inducing mast cell activation, 5 the Fc portion of IgM is superior at activation of the complement cascade, 6 while IgG can trigger ADCC. 3 Humans and the clinically relevant model organisms all possess Igs of the IgM, IgG, IgA and IgE classes (isotypes) as well as receptors for their Fc portions.Presumably, evolution selected for the maintenance of the different Ig isotypes and the Fc receptors speci c for them to maximize immune protection.
When considering the therapeutic mechanisms implicated in immunotherapy of cancer, both receptor blockade and Fc-mediated effector functions can play a role. 7,8In addition to the properties intrinsic to Igs, further functionalization, for example with cytotoxic drugs to create antibody-drug conjugates, can potentiate their therapeutic e cacy. 9The concept of checkpoint blockade has made the use of monoclonal antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the programmed cell death protein-1 and its ligand (PD-1/PD-L1) a cornerstone of cancer immunotherapy. 10,11These forms of immunotherapy rely on monoclonal antibodies, typically of the IgG class.This means that, for a given therapeutic antibody, only a single type of Fc portion determines its therapeutic e cacy.3][14][15][16][17] The situation is less clear-cut for interference with the PD-1/PD-L1 axis, where direct interference with the PD-1/PD-L1 interactions is believed to dominate over Fcmediated effects, but the latter has been documented as well. 18 have shown that nanobodies, the recombinantly expressed variable regions of heavy chain-only antibodies (VHHs), 19,20 can be used to target CTLA-4 and PD-L1. 16,21Perhaps not surprisingly, administration of nanobodies as immunotherapy is not particularly effective in the absence of an Fc portion.For CTLA-4, the installation of an Fc portion on such nanobodies is required for their antitumor properties. 16re we used a radically different approach to exploit Fc effector functions.We applied a strategy that enables engagement of all Ig isotypes, regardless of their speci city, to exploit the properties associated with the diverse Fc portions of all Igs (Fig. 1A).We showed that nanobodies that recognize Ig light chains (VHH kappa ) can be functionalized with small molecules to enforce recruitment to virus-infected cells of polyclonal Igs, regardless of isotype or speci city. 22We produced a conjugate of VHH kappa with the in uenza virus neuraminidase inhibitor zanamivir to eradicate cells infected with in uenza virus.These conjugates afforded protection against a challenge with a lethal dose of virus, even when given several days after infection.We found that zanamivir could be exchanged for a virus-speci c nanobody to yield a conjugate that likewise protected against in uenza infection.The surprising aspect of this nding is the complete lack of a requirement for antigen-speci c interactions of the recruited polyclonal Igs: all of the functional activity that eliminates virus-infected cells must be due to their Fc portions.The engagement of antibodies in this manner is conceptually similar to bi-speci c T cell engagers (BiTEs), bi-speci c constructs that typically comprise a module that recognizes the CD3 ε subunit of the TCR-CD3 complex, coupled to a tumor-speci c recognition module. 23,24Upon engagement by a BiTE, T cells, regardless of their speci city, are brought in contact with the target cell to be eliminated.When the BiTE is recruited to the tumor cell, it delivers an activation signal to the T cell to deploy its effector functions, such as cytotoxicity and cytokine release.
Here we extend the concept of nanobody-driven recruitment of polyclonal antibodies to tumor models (Fig. 1A).The circulatory half-life of VHH kappa conjugates is extended by binding to the much larger Igs, enabling stronger immune checkpoint blockade through sustained presence in the tumor microenvironment (TME).Mindful of the importance of Fc receptor engagement for e cacy of anti-CTLA-4 treatment, we wondered whether the recruitment of polyclonal Igs of all isotypes to CTLA-4 positive cells would improve antitumor activity when compared to a conventional monoclonal antibody.
We nd that this is indeed the case.We see a pronounced antitumor effect of the anti-CTLA-4 VHH-VHH kappa conjugate in the MC38 model of colorectal cancer, which we attribute primarily to depletion of regulatory T (Treg) cells in the TME.At the same time this treatment increases the number of T cells in the tumor.We made a similar conjugate of an anti-PD-L1 nanobody with VHH kappa .The anti-PD-L1 VHH-VHH kappa conjugate was less effective than the anti-CTLA-4 VHH-VHH kappa conjugate.Both types of conjugates still outperformed conventional monoclonal anti-CTLA-4 or anti-PD-L1 monoclonal antibodies in the MC38 model.To improve on the anti-PD-L1 VHH-VHH kappa conjugate, we created drug adducts of the anti-PD-L1 VHH-VHH kappa conjugate that were more effective than their unmodi ed counterparts (Fig. 1B).Both cytotoxic drugs and STING agonists increased antitumor activity when included in these conjugates.In the B16-F10/GVAX melanoma model, we found that both the anti-CTLA-4 VHH-VHH kappa and anti-PD-L1 VHH-VHH kappa conjugates were effective in controlling tumor growth and improving survival.These ndings introduce new possibilities for improvement of immunotherapies.

Production of bispeci c antibody engagers with retention of a nity and speci city
To arrive at the desired bispeci c antibody engagers, we produced bivalent nanobody conjugates (~ 30 kDa) in E. coli as genetic fusions of the anti-CTLA-4 VHH (H11) or the anti-PD-L1 VHH (A12) sequences with VHH kappa , respectively (Figure S1 and S2).Each conjugate carries a C-terminal LPETG sortase recognition motif, followed by a polyhistidine (6xHis) tag to allow puri cation on a nickel-nitrilotriacetic acid (Ni-NTA) matrix.Where indicated, the sortase reaction was used for site-speci c installation of triglycine-modi ed small molecule drug payloads at the C-terminus of the A12-VHH kappa conjugate (Fig. 1B) . 25The anti-CTLA-4 VHH H11 binds to murine CTLA-4, blocks its ligand-binding site and inhibits its interaction with B7-1, with stronger inhibitory activity than the anti-CTLA-4 monoclonal antibody 9H10. 16The anti-PD-L1 VHH A12 binds to murine PD-L1 with subnanomolar a nity and competes with the anti-PD-L1 monoclonal antibody 10F.9G2 for PD-L1 binding. 21 determine whether H11 and A12 retain their a nity and speci city for their respective targets after conjugation to VHH kappa , we generated saturation binding curves for both the free VHHs and the corresponding VHH kappa conjugates.Biotinylated H11-VHH kappa and A12-VHH kappa showed similar a nity for immobilized CTLA-4 and PD-L1, respectively, as biotinylated H11 and A12 (Fig. 2A).The VHH kappa portion of these conjugates binds to murine polyclonal IgG without loss of its subnanomolar a nity (Fig. 2B). 22The A12-VHH kappa conjugate recruited phycoerythrin (PE)-conjugated murine antibodies upon binding to PD-L1-positive B16-F10 cells.IgG, IgM, and IgA all bound to B16-F10 cells in the presence of the A12-VHH kappa conjugate but did not bind to PD-L1 knockout B16-F10 cells (Fig. 2C).
We evaluated the ability of the H11 and A12 VHHs to target cancer cells and tumor-in ltrating immune cells isolated from MC38 tumor-bearing C57BL/6 mice.Cyanine5 (Cy5)-labeled H11 binds to CTLA-4 on Tregs extracted from both tumor and spleen, with intratumoral Tregs showing higher CTLA-4 expression than those from the spleen (Fig. 2D).Similarly, A12-Cy5 binds to PD-L1 on wild-type MC38 cells (CD45 − ) and in ltrating CD11b + immune cells (CD45 + ) from both wild-type and PD-L1 knockout MC38 tumors (Fig. 2E).We conclude that the fusion proteins retain the speci city and a nity of their component parts.
The anti-CTLA-4 VHH (H11)-VHH kappa conjugate exhibits potent antitumor activity by depleting intratumoral regulatory T cells We examined the in vivo antitumor e cacy of the anti-CTLA-4 VHH (H11)-VHH kappa conjugate in the MC38 mouse colon carcinoma and B16-F10 melanoma models (Fig. 3A and 3B).For the MC38 model, mice received a subcutaneous injection of 1 × 10 6 cells.For the B16-F10 melanoma model, mice were injected subcutaneously with 5 × 10 5 B16-F10 melanoma cells and 5 × 10 5 irradiated B16-GVAX cells (dorsal and ventral, respectively).B16-GVAX cells produce granulocyte-macrophage colony-stimulating factor (GM-CSF) and serve as a vaccine when delivered at the time of tumor challenge. 26eatment consisted of three weekly injections over three weeks with 5 mg/kg of the tested VHHs, as indicated in the gures.The H11-VHH kappa conjugate slowed tumor growth and improved survival (Figs.3A and 3B, with individual tumor growth curves in Figures S3A and S3B), while a simple mixture of H11 and VHH kappa lacked antitumor activity.The H11-VHH kappa conjugate showed superior e cacy in the MC38 model compared to the anti-CTLA-4 mAb 9H10 (Fig. 3A) and showed similar e cacy in the B16-F10 melanoma model (Fig. 3B).The genetic fusion of SD36 to VHH kappa , which targets in uenza virus-infected cells, served as a speci city control and lacked antitumor activity (Figure S4).We installed click handles (an azide and a DBCO moiety) at the C-terminus of the two VHHs by sortase-catalyzed transpeptidation, yielding the genetically impossible C-to-C conjugate of H11-VHH kappa (Figure S5), which showed similar potent antitumor e cacy (Figure S6).The orientation of the VHHs (C to N, C to C) in such fusions is therefore not important for their antitumor properties.
3][14][15][16][17] We analyzed T cell populations recovered from the tumor after treatment with the H11-VHH kappa conjugate.We saw selective depletion of Tregs within TME (Fig. 3C, upper panels), without affecting Tregs in the draining lymph nodes or spleen.The percentage of total T cells as a fraction of all CD45 + cells increased in the TME of animals that received the H11-VHH kappa conjugate (Fig. 3C, lower panels).We examined the action of the H11-VHH kappa conjugate in mice de cient in complement component C3 or lacking the FcR common g chain to better distinguish between CDC and ADCC (Fig. 3D).Depletion of intratumoral Tregs still occurred in C3-de cient mice, but this depletion was compromised in FcR common g chain-de cient mice.Treg depletion by the H11-VHH kappa conjugate was thus attributable predominantly to FcgR-dependent cytotoxicity.
We next investigated the antitumor activity of the H11-VHH kappa conjugate on established MC38 tumors.
The half-life of the H11-VHH kappa conjugate was extended upon complex formation with the much larger Igs in the circulation (Fig. 3E).Therefore, treatment was set at 5 mg/kg of the VHHs or 12.5 mg/kg, an equimolar amount, of the anti-CTLA-4 antibody 9H10 administered on days 7, 10, and 13 post-tumor injection.The H11-VHH kappa conjugate outperformed 9H10 in inhibiting tumor growth and achieved 100% survival in mice bearing established tumors (Fig. 3F, with individual tumor growth curves in Figure S3C).An H11 conjugate with an albumin-speci c nanobody has been reported. 27This conjugate has a similar size and extended circulatory half-life (53 hours) as the H11-VHH kappa conjugate but lacks Fcdependent effector functions.Consequently, although the anti-albumin VHH-conjugated H11 demonstrated antitumor activity in the MC38 model, it required a much higher dose (30 mg/kg given intravenously) and a longer treatment period (3 weeks).Half-life extension is therefore not su cient for antitumor activity and requires Fc engagement for greater e cacy.The difference in antitumor activity between the VHH kappa -conjugated H11 and the anti-albumin VHH-conjugated H11 indicates that Fcmediated Treg depletion contributes to optimal antitumor activity.
The anti-PD-L1 VHH (A12)-VHH kappa conjugate accumulates in PD-L1-positive tumors PD-L1 is overexpressed by tumor cells and myeloid cells in the TME (Fig. 2E). 28To investigate whether the A12-VHH kappa conjugate speci cally accumulates in the tumor upon injection, we modi ed the conjugate with a near-infrared (NIR) dye (IRDye800CW) using a sortase reaction (Fig. 4A, S7, and S8) and performed NIR imaging.Using CRISPR/Cas9, we generated PD-L1 de cient MC38 and B16-F10 cell lines (Figure S9).Mice bearing wild-type and PD-L1 knockout MC38 tumors were injected intravenously with A12-VHH kappa -IRDye800CW on day 14 post-tumor injection.Imaging was performed 24 hours after injection of the imaging agents (Fig. 4B).A12-VHH kappa -IRDye800CW treated mice bearing wild-type MC38 tumors showed greater uptake of the NIR dye in tumors than other groups, including mice bearing PD-L1 de cient tumors (Fig. 4C and 4D).This uptake was blocked by a 30-fold molar excess of the unlabeled A12-VHH kappa conjugate.The uptake of A12-VHH kappa -IRDye800CW is therefore PD-L1dependent.The SD36-VHH kappa -IRDye800CW, which targets in uenza hemagglutinin and served as a speci city control, showed similar accumulation of dye as the other control groups.The much reduced uptake of A12-VHH kappa -IRDye800CW in the PD-L1 de cient tumors indicates that the MC38 tumor cells, rather than PD-L1-positive myeloid cells, are primarily responsible for the imaging agent's uptake.Biodistribution analysis showed that A12-VHH kappa -IRDye800CW speci cally accumulated in the tumor and not in other major organs (Fig. 4E), further supporting the suitability of the A12-VHH kappa conjugate for targeted delivery of various drugs to the tumor.

The antitumor activity of anti-PD-L1 VHH (A12)-VHH kappa requires PD-L1 expression by tumor cells
We examined the in vivo e cacy of the anti-PD-L1 VHH (A12)-VHH kappa conjugate using the MC38 mouse colon carcinoma and B16-F10 melanoma models (Fig. 5A and 5B), with the experimental setup described earlier.The A12-VHH kappa conjugate slowed tumor growth and improved survival (Fig. 5A and 5B, with individual tumor growth curves in Figures S10A and S10B), while a simple mixture of A12 and VHH kappa lacked antitumor activity.The A12-VHH kappa showed superior e cacy in both MC38 and B16-F10 models compared to the anti-PD-L1 mAb 10F.9G2 (Fig. 5A and 5B).The C-to-C conjugate of A12-VHH kappa also showed potent antitumor e cacy.(Figure S11 and S12).Unlike treatment with the anti-CTLA-4 VHH (H11)-VHH kappa conjugate, the A12-VHH kappa conjugate induced tumor rejection in only a fraction of the animals.We found that the half-life of the A12-VHH kappa conjugate was extended upon complex formation with circulating Igs (9.7 h, Fig. 5C), but to a lesser extent than that of the H11-VHH kappa conjugate (44.2 h, Fig. 3E).We implanted MC38 wild-type and MC38 PD-L1 knockout cells in C57BL/6 mice and started treatment at day 1 post-injection.Mice bearing tumors lacking PD-L1 failed to respond to treatment (Fig. 5D and S10C).Thus, PD-L1 expression by tumor cells is required for the e cacy of the A12-VHH kappa conjugate treatment.

Improving the antitumor effect of the anti-PD-L1 VHH (A12)-VHH kappa conjugate through production of cytotoxic drug conjugates
In view of the limited antitumor activity of the A12-VHH kappa conjugate we generated a A12-VHH kappa drug adduct.We rst functionalized the A12-VHH kappa conjugate with maytansine, a cytotoxic drug that binds to tubulin and inhibits microtubule assembly, 29 for targeted delivery of this toxin to the TME.Two triglycine-modi ed maytansine derivatives, DM1 (with a non-cleavable linker, Figure S13) and DM4 (with a disul de cleavable linker, Figure S14), were site-speci cally conjugated to the C-terminus of the A12-VHH kappa conjugate using a sortase reaction (Figure S15 and S16).Although B16-F10 murine melanoma cells were less sensitive to maytansine in vitro (EC 50 : 47.7 nM) compared to EL4 murine T cell lymphoma cells reported previously (EC 50 : 3.9 nM), 30 the DM1-modi ed A12-VHH kappa showed comparable cytotoxicity (EC 50 : 48.1 nM) as free maytansine after a three-day incubation with B16-F10 cells with high PD-L1 expression (Figure S17A).This effect is approximately three times stronger than that seen for SD36-VHH kappa -DM1, used as a speci city control.In contrast, when tested on PD-L1 de cient B16-F10 cells, the cytotoxicity of A12-VHH kappa -DM1 was reduced to a level similar to that of SD36-VHH kappa -DM1 (Figure S17A), con rming that the increased uptake of A12-VHH kappa -DM1 depends on surfaceexpressed PD-L1.The DM4-modi ed VHHs were also cytotoxic, similar to the DM1-modi ed VHHs (Figure S17B).As expected, the absolute potency of the conjugates decreased when the incubation time with the cells was reduced from 3 days to 2 hours, a duration chosen to prevent the extracellular reduction of disul de bonds in the DM4-modi ed VHHs.
Both DM1-and DM4-modi ed A12-VHH kappa conjugates have antitumor activity in the MC38 models, with the DM4-modi ed conjugate achieving 100% survival.(Fig. 6A and S18A).We used the DM4modi ed VHHs for further experiments.Treatment with the A12-VHH kappa conjugate seven days after MC38 cell implantation delayed tumor growth but did not signi cantly improve the survival rate (Fig. 6B  and S18B).In contrast, 5 out of 6 mice treated with A12-VHH kappa -DM4 survived and showed negligible tumor growth.Neither the speci city control VHH (SD36)-VHH kappa -DM4 nor A12-DM4 prevented tumor growth.No weight loss was observed in any of the mice that received the conjugates, suggesting a lack of overt toxicity of the DM4 adducts at the given doses (Fig. 6C).Most of the cells harvested from the TME after two doses of A12-VHH kappa -DM4 were non-viable (Fig. 6D).The percentage of CD11b + myeloid cells among the in ltrating immune cells was reduced (Fig. 6D).We tested the antitumor activity of A12-VHH kappa -DM4 on the more aggressive B16-F10 melanoma model in the absence of the GVAX vaccine (Figs.6E and S18C).We found that modi cation of the A12-VHH kappa conjugate with DM4 enhanced its potency against tumor growth and improved survival.

Targeted delivery of a STING agonist enhances antitumor activity of the anti-PD-L1 VHH (A12)-VHH kappa conjugate
To facilitate the establishment of an in ammatory environment conducive to an antitumor immune response, 31 we next created adducts that include a STING agonist. 32Targeted delivery of a cyclic dinucleotide STING agonist by means of further modi cation of the A12-VHH kappa conjugate (Figure S19) to the TME activated the STING pathway.Treatment with A12-VHH kappa -STING agonist signi cantly suppressed the growth of MC38 tumors and cured 50% of the mice, demonstrating greater potency than the simple combination of the A12-VHH kappa conjugate and an equimolar amount of free STING agonist (Fig. 7A and S20A).The lack of signi cant body weight loss induced by the A12-VHH kappa -STING agonist suggests that this conjugate does not cause systemic in ammation (Fig. 7B).The percentage of activated (CD69-positive) CD4 + and CD8 + T cells harvested from the tumors of mice treated with two doses of A12-VHH kappa -STING agonist increased signi cantly (Fig. 7C), indicating that the STING agonist enhances the generation of a T cell response, including the induction of CD8 + cytotoxic T cells.The A12-VHH kappa -STING agonist also reduced the growth of the more aggressive B16-F10 tumor more effectively than the A12-VHH kappa conjugate, but it did not improve the overall survival (Fig. 7D and   S20B).Increasing the dose of A12-VHH kappa -STING agonist from 5 mg/kg to 10 mg/kg showed stronger inhibition of tumor growth and improved survival, but with a slight increase in body weight loss (Fig. 7E  and S20C).

Discussion
Bi-speci c T cell engagers (BiTes) are in clinical use as antitumor agents.Dual speci city is achieved through the creation of fusions of a T cell recognition module, typically anti-CD3ε, and an antitumor component.The success of BiTes derives from their ability to direct T cells, regardless of their speci city, to the tumor cell and exploit their effector functions. 23,24Here we extend the concept of bi-speci c engagers by exploiting the ability of fusion proteins to target cells relevant to the antitumor response, and harness the effector functions of all Ig classes in the absence of deliberate immunization.
Nanobodies and their derivatives have advantages over the use of conventional antibodies, including: (1)   high-yield expression including in prokaryotic expression systems due to their smaller size, superior stability and solubility, as well a lesser dependency on disul de bond formation and glycosylation; (2) the ability to access epitopes less accessible to conventional antibodies, facilitated by their extended complementarity-determining region 3 (CDR3) loop; (3) low immunogenicity owing to their sequence homology to human immunoglobulin (Ig) V regions and humanization strategies; and (4) their small size and ease of modi cation, allowing for con gurations that are more challenging to achieve with conventional antibodies. 20,33,34However, when used as stand-alone therapeutic agents, nanobodies suffer from a short circulatory half-life (< 30 minutes in mice and 0.5-2 hours in humans). 35,36nobodies obviously lack Fc-dependent effector functions.To overcome the latter shortcomings, a widely used strategy is to conjugate the nanobody of interest with an albumin-speci c nanobody. 37,38hese conjugates extend their half-life by binding to serum albumin in the bloodstream but cannot exert Fc-dependent effector functions.Alternatively, conjugating the nanobody to the Fc fragment can address the lack of Fc-dependent functions, though it compromises some of the nanobody's desirable properties. 39The approach described here achieves both circulatory half-life extension and provides Fcdependent effector functions, not limited to a single Ig isotype.
Adult mice have the peculiar trait, in that > 95% of all Ig isotypes carry the kappa light chain, 40 possibly driven by the large number of V k genes that provide the substrate for selection of B cells of the requisite speci city and a l light chain locus of much reduced complexity.In humans, the percentage of circulating Igs carrying kappa light chains is ~ 60%. 41We do not anticipate this difference to impact the effectiveness of mouse vs. human VHH kappa conjugates signi cantly.Given the quantities of circulating Igs in humans and mice, as well as the doses of VHH kappa conjugates administered, only a small fraction of the circulating Igs will bind the conjugates.The availability of a nanobody speci c for human Ig kappa light chains with picomolar binding a nity suggests the potential for using the VHH kappa conjugate strategy in humans. 42 show improved activity of the H11-VHH kappa conjugate over 9H10 monoclonal antibody in two tumor models.We attribute this improvement to the engagement of all Fc-associated functions, rather than the reliance on a single Fc portion.MC38 is notoriously unresponsive to CTLA-4 blockade, 43,44 yet all the H11-VHH kappa conjugate-treated mice successfully rejected tumors.Using H11-Cy5, we con rmed higher levels of CTLA-4 expression on intratumoral Tregs than on splenic Tregs (Fig. 2D) as a possible mechanism for the tumor-speci c depletion of Tregs. 45Using FcgR-de cient and complement-de cient mice, we demonstrate that Treg depletion is mediated by FcgR.The fraction of Tregs in the spleen increased upon treatment with the H11-VHH kappa conjugate (Fig. 3C).This was still the case in FcgRde cient and complement-de cient mice (Fig. 3D), suggesting an Fc-independent mechanism resulting from CTLA-4 signal blockade itself.Our data suggest that polyclonal Ig recruitment and harnessing the diversity of Fc-mediated effects could improve activity of CTLA-4 blockade in human.In MC38 and B16-F10 models, the anti-PD-L1 A12-VHH kappa conjugate outperformed the reference 10F.9G2 monoclonal antibody but failed to confer complete protection.Flow cytometry of established tumors showed that PD-L1 is expressed on both MC38 tumor cells and tumor-in ltrating CD11b + myeloid cells (Fig. 2E).Because PD-L1 knockout MC38 tumor-bearing mice failed to respond to the A12-VHH kappa conjugate therapy, expression of PD-L1 on tumor cells is required for therapeutic e cacy, supported by the imaging data (Fig. 4).
We hypothesize that depletion of tumor-associated macrophages in the MC38 model is key to initiate tumor rejection.To improve on the suboptimal activity of the A12-VHH kappa conjugate, we incorporated a cytotoxic drug, DM4.Treatment with A12-VHH kappa -DM4 improved tumor rejection in both the MC38 and B16-F10 models.MC38 tumors treated with A12-VHH kappa -DM4 contained few live cells and had fewer CD11b + CD45 + myeloid cells (Fig. 6D), suggesting that A12-VHH kappa -DM4 increased the depletion of tumor-associated macrophages.Delivery of pro-in ammatory signals to the TME promotes tumor clearance. 46,47Site-speci c incorporation of a STING agonist in the A12-VHH kappa conjugate improved rejection of MC38 tumors.A12-VHH kappa STING agonist induced activation of CD8 + and CD4 T + cells (Fig. 7C).Incorporation of the STING agonist in the A12-VHH kappa conjugate delayed tumor growth in the B16-F10 model but failed to improve overall survival.
The greater antitumor activity of the VHH kappa conjugates when compared with the widely used anti-CTLA-4 monoclonal antibody suggests that different FcR + cells may be responsible for this effect.The contribution of different FcR + cells in response to deployment of the VHH kappa conjugates requires further exploration.Given the conservation of multiple Ig isotypes across vertebrate species, there may well be an advantage to the engagement of multiple classes of Igs over a single one.We compared the e cacy of monoclonal antibodies speci c for CTLA-4 or PD-L1 with the corresponding VHH kappa conjugates and found the latter to be superior.Perhaps other monoclonal antibodies would perform better than those used here, which have been widely used preclinically, but we consider our results as supporting the value of multiple Ig engagement.
The VHH kappa -nanobody approach can be extended to target additional cancer-speci c markers.
Eradication of CD20-, HER2-, or EGFR-positive cancer cells by therapeutic antibodies is driven by ADCC/CDC as well as ADCP. 7Immunomodulatory reagents can deplete speci c immunosuppressive cells to enhance antitumor activities of the remaining T cells.Appealing targets for immunomodulation by VHH kappa conjugates include OX40, CD25, GITR, CD47 and CD73. 7,8,48Our ndings support the pursuit of bispeci c antibody engagers to target additional cancer-speci c and immunomodulatory markers.

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